general article neuropeptides: the slower neurotransmitters

GENERAL ARTICLE

Neuropeptides: The Slower Neurotransmitters∗

Umer Saleem Bhat and Kavita Babu

Umer Saleem Bhat received

his MSc in Biotechnology

from the University of

Kashmir and is currently a

graduate student at IISER

Mohali. Umer is studying the

function of neuropeptides in

C. elegans for his PhD thesis

research.

Kavita Babu holds a PhD in

developmental biology from

The National University of

Singapore. She has worked

on C. elegans at

Massachusetts General

Hospital for her postdoctoral

research. Kavita headed her

lab at IISER Mohali for close

to eight years before moving

to the Centre for

Neuroscience, IISc in 2019.

Her lab is largely interested

in understanding the

molecular mechanisms of

synaptic function.

Neuropeptides, as the name suggests, are small peptides re-

leased by neurons that allow them to communicate with each

other. These small peptides modulate the activity of neurons,

in turn, allowing the modulation of multiple behaviors. Here,

we describe how neuropeptides are made and go on to dis-

cuss how these peptides function in an organism. We also

highlight the specific roles of neuropeptides in modulating the

locomotory behavior of the free-living nematode, Caenorhab-

ditis elegans.

Introduction

Cell signaling refers to the process by which cells communicate

with each other and their environment to modulate different cel-

lular functions. Cells in our body secrete a plethora of molecules

known as the signaling molecules. These molecules are required

for the transduction of information from one cell to another. Neu-

ropeptides are small peptides that are secreted by neurons, al-

lowing them to ‘talk’ to each other. These peptides bind to the

surface receptors on the postsynaptic neurons or extra synapti-

cally (in an effector cell not in physical contact with the pep-

tide producing neuron) and allow for neuronal activation or in-

activation. Different populations of our brain cells release mul-

tiple sets of these peptides. Neuropeptides have been found to

be involved in regulating a wide range of functions, including

learning and memory, locomotion, food intake, social behavior,

reproduction, metabolism, reward behavior, analgesia, etc. Fur-

ther, defective neuropeptide signaling is related to various neu-

rological diseases like autism, Alzheimer’s disease, and epilepsy.

∗Vol.25, No.12, DOI: https://doi.org/10.1007/s12045-020-1094-8

RESONANCE | December 2020 1741

GENERAL ARTICLE

However, the mechanism of how these peptides function is stillKeywords

Neuropeptides, receptors, and C.

elegans.

largely unknown. In this short review, we first discuss neuropep-

tide biology in general and then provide a mechanistic insight into

neuropeptide control of locomotion behavior of the small free-

living nematode, Caenorhabditis elegans, under normal well-fed

or starvation-induced stress conditions. The C. elegans nervous

system releases more than a hundred neuropeptides, some of which

have mammalian counterparts. More importantly, the mechanism

of function of neuropeptides is conserved, from worms to verte-

brates, including Homo sapiens.

Neuropeptide Processing: From Precursor to Functional Ma-

turity

FunctionallyFunctionally mature

neuropeptides are

derived from large

precursor molecules

after a series of

processing and

modification steps. A

single neuropeptide or

multiple distinct

neuropeptides can be

derived from a single

precursor molecule.

mature neuropeptides are derived from large precur-

sor molecules after a series of processing and modification steps.

A single neuropeptide or multiple distinct neuropeptides can be

derived from a single precursor molecule. However, in mammals,

a single precursor molecule can be subjected to differential cleav-

age to yield a different set of neuropeptides. These neuropeptide

precursors are synthesized on the endoplasmic reticulum (ER) in-

side the soma of the neuron and are called pre-propeptides. The

peptide signal sequence that indicates to the cell that the protein

must be secreted is first cleaved inside the ER yielding a propep-

tide. The propeptide then traverses the Golgi apparatus where it

is packaged into vesicles. The final processing and modification

of the propeptide to yield the mature neuropeptide takes place in-

side the vesicle (illustrated in Figure 1). The series of events and

enzymes involved in processing and modification of the propep-

tide to yield a mature neuropeptide are shown in Figure 2. The

processing of the propeptide begins with the action of an endopro-

teolytic kex2/subtilisin-like proprotein convertase. The endopro-

teolytic cleavage generally takes place at the C-terminal dibasic

residues like Lys-Arg, Arg-Arg, Lys-Lys, and Arg-Lys, flanking

the peptide sequence. Reports have confirmed that cleavage could

also happen at the monobasic and tribasic residues in specific sce-

narios.

1742 RESONANCE | December 2020

GENERAL ARTICLE

Figure 1. Neuropeptide

biosynthesis, processing,

and storage pathway. Neu-

ropeptide precursors, after

being transcribed, are syn-

thesized as pre-propeptides

on the ribosomes at the

rough endoplasmic reticu-

lum (1) Propeptides along

with the processing en-

zymes from the ER traverse

to the Golgi apparatus (2)

here, the processing of

neuropeptides starts in the

vesicles after which they are

transported along the axon

(3) to the axon terminals

with the help of motor pro-

teins (green lines and (4)).

Neuropeptides are stored

inside large dense-core

vesicles (5) and after being

released they diffuse away

to act on cells that could be

at a distance from the cell

releasing the neuropeptide

(6). Source: Modified from

Basic Neurochemistry, 6th

ed, 1999. Lippincott-Raven.

C.elegans as the model organism for the study of molecular mech-

anisms in the nervous system is ideal as it has just 300 well-

characterized neurons, compared to approximately 86 billion neu-

rons in the human brain. It is also easy to do genetic manipula-

tions to study the effect of mutations on these worms. C. elegans

express four types of propeptide convertases including KPC-1,

EGL-3/KPC-2, AEX-5/ KPC-3, and BLI-4/KPC-4. These en-

zymes have different targets and preferences depending upon their

catalytic domains. The cleavage of propeptides by the action of

propeptide convertase is followed by the removal of the basic

residues from the C-terminus of the intermediate cleaved prod-

ucts. The enzyme that is responsible for the removal of basic

residues is known as carboxypeptidase E (CPE).

Even after the endoproteolytic and exoproteolytic cleavage of the

precursor molecules, they are biologically inactive and suscepti-

ble to degradation. For the peptide to be biologically active, mod-

ifications at the C-terminus and, in some cases, the N-terminus

must take place. ‘Amidation’ is the most common modification of

inactive neuropeptides based on the presence of a glycine residue

at the C-terminus, which donates an amide group during this pro-

cess. The process of amidation is catalyzed by a bifunctional

enzyme known as peptidylglycine α-amidating monooxygenase

(PAM). The structural analysis of PAM has revealed two domains,

which sequentially catalyze the two-step process of amidation,

respectively. The first reaction involves the conversion of pep-


GENERAL ARTICLE

Figure 2. Processing of

pre-propeptide to yield the

mature neuropeptide. Pre-

cursor molecules having sig-

nal peptides are first sub-

jected to the action of signal

peptidase, which cleaves the

signal peptide. The propep-

tide thus formed is acted

upon by the enzyme pro-

tein convertase at mono/di-

basic residues yielding small

peptide sequences. Basic

residues are removed from

these peptide sequences by

carboxypeptidase E. Finally,

post-translational modifica-

tions like amidation occur

at the C-terminal glycine

residue yielding a mature

neuropeptide. Source: Mod-

ified from C Li and K Kim,

Neuropeptides, WormBook,

1–36, 2008.

tidylglycine into peptidyl α-hydroxyglycine, and is catalyzed by

the peptidylglycine α-hydroxylating monooxygenase (PHM) do-

main of PAM. The intermediate product of the first reaction serves

as the substrate for the second reaction, and is acted upon by

peptidyl-α-hydroxyglycine α-amidating lyase (PAL), yielding the

final product in the form of an amidated peptide and glyoxylate

as a by-product (illustrated in Figure 3).

Neuropeptide Release

AsThe processing of

neuropeptides begins in

the secretory vesicles

from the trans-Golgi

network. The bioactive

neuropeptides are then

stored in the same

vesicles known as large

dense-core vesicles.

discussed in the previous section, the processing of neuropep-

tides begins in the secretory vesicles from the trans-Golgi net-

work. The bioactive neuropeptides are then stored in the same

vesicles known as large dense-core vesicles, in contrast to the

small clear-core vesicles that store conventional small neurotrans-

mitters. Unlike small clear-core vesicles that are localized at the

presynaptic specialization, the large dense-core vesicle contain-


GENERAL ARTICLE

Figure 3. Sequential re-

action of bifunctional PAM

catalyzed by PHM and PAL.

Source: Modified from Chu-

fan, et al., 1993.

ing neuropeptides are scattered at the nerve terminus (illustrated

in Figure 1). A variety of neurons have been found to show the

vesicular release of neuropeptides from the cell body and den-

drites as well. Neurotransmitters are secreted in response to the

neural activation, i.e., the arrival of a nerve impulse that causes

an increase in the intracellular levels of calcium due to influx of

calcium ions through the voltage-gated calcium (Ca+2) channels.

Calcium also helps in the exocytosis of the vesicles by docking

it to the plasma membrane, and thereby, leading to the release of

neurotransmitters. The release of small neurotransmitters occurs

in the proximity of transmembrane Ca+2 channels. However, the

calcium that leads to the exocytosis of vesicles containing neu-

ropeptides may also come from the intracellular calcium stores of

the transmembrane calcium influx. Once the neuropeptides Neuropeptides are

involved in diverse

physiological functions.

Several reports have

shown that a single

neuropeptide is

responsible for

regulating more than one

physiological process.

are

released from the neurons, there is no reuptake mechanism for

these peptides, as is the case with small neurotransmitters. En-

dogeneous neuropeptides can, however, be maintained at physio-

logical levels, as these peptides can be degraded by extracellular

proteases.

Mechanism of Action: Slow Neurotransmission

Neuropeptides 1Neuropeptides showing

pleiotropic effects implies that

loss of a single neuropeptide

could give rise to multiple

unrelated phenotypes.

are involved in diverse physiological functions.

Several reports have shown that a single neuropeptide is respon-

sible for regulating more than one physiological process. Hence,

many neuropeptides are thought to have pleiotropic1 effects. Neu-

ropeptides, after being released from their source neurons diffuse


GENERAL ARTICLE

and act at relatively large distances. This process of dispersion

by diffusion is known as ‘volumetric transmission’. UnlikeUnlike conventional

small chemical

neurotransmitters,

neuropeptides perform

their function slowly for

a longer duration. Small

neurotransmitters are

either degraded in the

synaptic cleft (e.g.

acetylcholinesterase

hydrolyzes

acetylcholine) or are

transported back by

endocytosis after they

have performed their

function at the synapse.

However, till date, no

such reuptake machinery

for neuropeptides has

been reported.

con-

ventional small chemical neurotransmitters, neuropeptides per-

form their function slowly for a longer duration. Small neuro-

transmitters are either degraded in the synaptic cleft (e.g. acetyl-

cholinesterase hydrolyzes acetylcholine) or are transported back

by endocytosis after they have performed their function at the

synapse. However, till date, no such reuptake machinery for neu-

ropeptides has been reported. Volumetric transmission and lack

of reuptake machinery contribute to the long-lasting effects of

neuropeptides. Neuropeptides exert their function by binding to

their respective G-protein coupled receptors (GPCRs) and thus al-

ter the levels of second messengers in the effector cells. This sig-

nal cascade leads to several changes in the cells and affects gene

expression to modulate various processes like behavior, synapto-

genesis, cellular morphology, trafficking, etc. Unlike small neu-

rotransmitters, which bind to their receptors in the postsynaptic

cell at the synaptic cleft, neuropeptides can bind to their receptors

extrasynaptically, on effector cells that are not physically con-

nected to the peptide producing neurons through synapses but in-

stead might be spatially separated over a large distance. However,

there are also instances where neuropeptides bind to the recep-

tors on the postsynaptic cell, like in the case of neuropeptide Y

which is released from the arcuate nucleus in the hypothalamus,

binds to its receptor on the postsynaptic pro-opiomelanocortin

(POMC) neuron, and goes on to hyperpolarize this neuron. Due

to the pleiotropic effect of neuropeptides, a single neuropeptide

can bind to more than one kind of GPCR, and one GPCR can also

respond to more than one type of neuropeptide ligand. Therefore,

it is very difficult to discern the exact mechanism of neuropeptide

functioning in a neural circuit.

The Functioning of the FLP-18 Neuropeptide

Our lab is interested in delineating the role of neuropeptides and

the molecular mechanism by which these peptides carry out the

signaling process to regulate behavior in the nematode Caenorhab-


GENERAL ARTICLE

ditis elegans. So far in C. elegans, 113 neuropeptide genes have

been identified which code for over 250 distinct neuropeptides.

Of these genes, 31 genes code for FMRFamide-related peptides

(FLP), 40 genes code for insulin-like peptides (INS), and 42 genes

encode non-insulin, non-FMRFamide-related neuropeptides (NLP).

A recent report from our lab has shown that the FLP-18 neuropep-

tide is involved in regulating the reversal length2 2Reversal length is the distance

that the C. elegans moves back-

wards by before changing di-

rection.

in C. elegans

through its GPCRs, NPR-1 and NPR-4. The locomotory behav-

ior of C. elegans is characterized by forward movement with oc-

casional pauses followed by reversing back in order to change

its direction during the navigation (illustrated in Figure 4). Re-

versal is an important survival strategy for C. elegans, allowing

it to explore its environment in search of food and also to avoid

being the target of predators or any noxious stimulus. In particu-

lar, the worm shows a very interesting behavior while it searches

for food under laboratory conditions. When When the well-fed worm

is put on a plate without

food, it searches for food

in its proximity for

approximately 15

minutes. This behavior,

known as ‘local search’,

is characterized by

frequent reversals and

reorientations as one can

imagine the worm to do

in order to look for food

in its immediate

surroundings.

the well-fed worm

is put on a plate without food, it searches for food in its prox-

imity for approximately 15 minutes. This behavior, known as

‘local search’, is characterized by frequent reversals and reori-

entations as one can imagine the worm to do in order to look

for food in its immediate surroundings. These reversals help the

worm change direction quite frequently, and as a result, there is

very little dispersion from the point on the plate where the worm

was initially placed. If the worm fails to encounter food during

its local search, it shifts gears to go into ‘global search’. During

the global search, the reversal circuitry is suppressed and as a re-

sult, the number of reversals go down significantly. Decreased

reversals help the worm move without changing its direction so

that it can explore more areas as compared to the local search

area. The length of reversals in each exploratory behavior is cru-

cial for C. elegans as the reversal length plays an important role

in determining the angle by which the worm will change its di-

rection. The longer the reversal, the larger will be the angle (il-

lustrated in Figure 4). During a local search, the reversals are

longer, and consequently, the direction change angles would also

be greater as compared to those during the global search. Local


GENERAL ARTICLE

search behavior is characterized by frequent and long reversals,

which enables the worm to reorient itself and look for food in

close vicinity. Hence, it can be speculated that the sum totals

of reversal frequency and the reversal length result in the stereo-

typic exploration during a local search. However, the length of

reversal is a critical and determining factor for the reorientation

angle. A report from our lab has shown that FLP-18 acts like a

switch and negatively regulates the reversal length. FLP-18, af-

ter being released from the AVA33Note that worm neurons are

named based on their position.

neuron, binds to its receptors

on the target neuron ASE and AVA itself to suppress the circuitry

controlling the length of reversals. In worms lacking flp-18, the

reversals during local search behavior are longer as compared to

those seen in wild type worms. Moreover, worms mutant for the

FLP-18 receptors npr-1 and npr-4 show a similar phenotype to

that of flp-18 mutants, which confirms that FLP-18 is the ligand

for NPR-1 and NPR-4. The inhibitory action of FLP-18 was fur-

ther confirmed by calcium imaging of the AVA neuron, which is

responsible for the reversals in C. elegans. Calcium is released

inside the neuron when it receives the impulse signal and is set

to fire. A rise in intracellular calcium indicates that the neuron is

firing and is active to perform its function. CalciumCalcium imaging is a

microscopic technique in

which calcium

dependent fluorescent

protein (GCaMP) is

expressed specifically in

the target neuron using a

neuron-specific

promoter.

imaging is

a microscopic technique in which calcium depending fluorescent

protein (GCaMP) is expressed specifically in the target neuron

using a neuron-specific promoter. A rise in intracellular calcium

is indicated by a change in the fluorescence activity of GCaMP.

In flp-18 mutant worms, increased calcium levels were observed

in the AVA neuron during reversals as compared to the wild type

worms. This indicated that the presence of FLP-18 suppresses

the AVA activity and thus controls the reversal length. Our lab

has also shown that there is a decrease in the expression of FLP-

18 during the local search, as it can be very well correlated with

the fact that the worm needs to perform longer reversals and thus

more reorientations, allowing the worm to perform local search

properly. In contrast, the expression of FLP-18 increases 20 folds

during the global search, and this increased expression attenuates

the reversal length circuitry as already discussed, thus decreasing

the length of reversals and reorientations and allowing the worm


GENERAL ARTICLE

Figure 4. Illustrations of

different movements in C.

elegans. The four images

show forward straight, for-

ward curved, turn and rever-

sals respectively. In the last

illustration of reversal and

turn, the C. elegans moves

from position 1 to 2, then

makes a reversal to point 3

and then turns at an angle (θ)

and moves towards points 4

and 5. If the reversal is

longer, the angle of turn af-

ter the reversal increases.

to transition into global search. This study has shed light on the

importance of neuropeptides in regulating multiple behaviors of

an organism. Hence, we can speculate that any defect in the ex-

pression, synthesis, timing of the release, or binding of these neu-

ropeptides could lead to severe disease conditions in an organism.

Neuropeptides and Diseases: It Is the Balance that Matters

Neuropeptide signaling in our brain has been associated with nu-

merous physiological functions. However, the implication of these

neuropeptides in the pathophysiology of several brain-related dis-

eases is yet to be completely understood. Several reports indicate

that alteration in peptidergic signaling leads to severe pathology

in the brain. For instance, aging-related neurodegeneration has

been linked with the loss or reduction in peptidergic signaling in

the brain. Multiple studies have confirmed the altered concentra-

tion of several neuropeptides in the cerebrospinal fluid and brain


GENERAL ARTICLE

tissues in patients with pathologies like dementia. Here, weMultiple studies have

confirmed the altered

concentration of several

neuropeptides in the

cerebrospinal fluid and

brain tissues in patients

with pathologies like

dementia.

dis-

cuss some of the diseases that are associated with alterations in

the levels of neuropeptides.

One of the most common neurodegenerative diseases is Alzheimer’s

disease (AD). It is a progressive disease associated with the loss

of neurons, which leads to severe conditions like dementia. In

Alzheimer’s disease-related dementia, levels of the neuropeptide

somatostatin are reduced as compared to the normal brain tissues.

Somatostatin plays a critical role in neurotransmission and cell

proliferation. Loss of somatostatin is believed to cause the de-

generation of somatostatin neurons, which leads to the loss of

normal signaling cascade and hence the neurodegenerative dis-

ease. AD has also been linked with ghrelin. Ghrelin is a 28 amino

acid long, orexigenic neuropeptide, involved in numerous physi-

ological functions including neuroprotective functions, neuroen-

docrine secretion, energy homeostasis, and higher brain functions

like mood, reward related behaviors, and learning and memory.

Reduced mRNA levels of ghrelin have been reported in the brain

tissues of patients with AD. Also, one of the single nucleotide

polymorphisms of the ghrelin gene rs4684677 (Leu90Gln) is as-

sociated with the onset of Alzheimer’s disease.

As already discussed, ghrelin is involved in modulating several

higher brain functions. Hence, it is unsurprising that altered levels

of this peptide critically affect the balance of neuronal signaling.

Apart from AD, low levels of ghrelin in the brain have also been

associated with Parkinson’s disease (PD). PD is also one of the

most common neurodegenerative diseases affecting locomotion

due to a drop in dopamine levels in the brain.

Neuropeptides signaling plays a vital role in regulating metabolic

homeostasis. There is a tight regulation of orexigenic and anorex-

igenic neuropeptide signaling with respect to the metabolic status

of the organism. Orexigenic neuropeptides include those that are

involved in appetite stimulation and consequently relay signals

to increase food intake and reduce energy expenditure. Ghrelin,

neuropeptide Y, and orexin are well-studied orexigenic neuropep-

tides. On the other hand, anorexigenic neuropeptides are those


GENERAL ARTICLE

that act as appetite suppressors, inhibiting food intake, and en-

hancing energy expenditure. Leptin is a vital anorexigenic neu-

ropeptide. Any defect in the precise balance of these neuropep-

tides can thus lead to metabolic syndromes. For instance, an in-

crease in neuropeptide Y inhibits POMC neurons in the hypotha-

lamus. POMC neurons sense the metabolic status of the organism

and maintain the glucose and energy homeostasis levels. Thus,

inhibition of POMC neurons due to upregulation of neuropeptide

Y leads to increased appetite stimulation and less expenditure of

energy. This is thought to be one of the major causes of early

onset of obesity.

Conclusion

Neuropeptides are small peptides secreted by neurons or other

cells that act as signaling molecules in the nervous system. These

are slow neurotransmitters that primarily bind extra synaptically

to one or more specific GPCRs and relay signals by changing the

levels of second messengers. These peptides produce sustained

effects as there is no reuptake machinery, which can clear these

molecules from the synapse. Neuropeptide signaling modulates

multiple physiological processes and higher-order brain functions

like cognition, learning, memory, locomotion metabolism, and

the likes. Any alteration in the levels of these peptides could

lead to severe pathologies like Alzheimer’s disease and various

metabolic disorders. It can be concluded that elucidating the elu-

sive mechanism of action of these molecules might be impor-

tant not only for better understanding the physiological states of

organisms but also to identify novel biomarkers for debilitating

pathophysiological states.

Suggested Reading

[1] C Li and K Kim, Neuropeptides, WormBook, pp.1–36, 2008.

[2] Basic Neurochemistry, 6th ed (Molecular, Cellular and Medical Aspects), 1999;

Lippincott-Raven.


GENERAL ARTICLE

[3] E E Chufan, M De, B A Eipper, R E Mains and L M Amzel, Amidation of

bioactive peptides: The structure of the lyase domain of the amidating enzyme,

Structure, Vol.17, No.7, pp.965–973, 2009.

Address for Correspondence

Umer Saleem Bhat

Department of Biological

Sciences

Indian Institute of Science

Education and Research

IISER Mohali

Knowledge City

Sector 81, SAS Nagar

Manauli PO 140 306, Punjab,

India.

Email: [email protected]

Kavita Babu

Centre for Neuroscience

Indian Institute of Science

C V Raman Road

Bangalore 560 012, India.

Email:

[email protected]

[4] A Bhardwaj, S Thapliyal, Y Dahiya and K Babu, FLP-18 functions through the

G-protein-coupled receptors NPR-1 and NPR-4 to modulate reversal length in

Caenorhabditis elegans, J. Neurosci., Vol.38, No.20, pp.4641–4654, 2018.

[5] M F Beal and J B Martin, Neuropeptides in neurological disease, Ann Neurol.,

Vol.20, No.5, pp.547–565, 1986.

[6] A A van der Klaauw, Neuropeptides in obesity and metabolic disease, Clinical

Chemistry, Vol.64, No.1, pp.173–182, 2018.

[7] N Shibata, T Ohnuma, B Kuerban, M Komatsu and H Arai, Genetic associ-

ation between ghrelin polymorphisms and Alzheimer’s disease in a Japanese

population, Dement Geriatr Cogn Disord., Vol.32, No.3, pp.178–181, 2011.


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